The Hedgehog (HH) signaling pathway is vital for tissue patterning and organ formation during embryogenesis, as well as adult tissue homeostasis, renewal and regeneration. In contrast, aberrant HH pathway function results in numerous developmental diseases and birth defects, and is responsible for a growing number of cancers. GLI proteins (GLI1-3) are essential downstream transcriptional effectors of the HH pathway, with both transcriptional activator and repressor functions. Recent work indicates that primary cilia are essential regulators of HH pathway activity, in part through the ciliary targetin and trafficking of GLI proteins. There are critical gaps, however, in our understanding of the mechanisms by which these proteins traffic through cilia, and the consequences that this trafficking has on GLI protein function downstream of HH pathway activation. Here we propose to answer the following questions: what regulates the ciliary targeting and trafficking of GLI proteins, and how does this impact GLI transcriptional activity? The long-term goal of this research is to understand how ciliary transport of HH pathway components affects their function during HH signal transduction in development and disease. The objective of this proposal is to precisely define the mechanisms by which the GLI proteins traffic through cilia and the functional consequences of this transport on GLI-mediated HH signaling. Our data indicate that GLI proteins selectively interact with members of the kinesin-2 family of motor proteins, and that disrupting these interactions significantly impacts GLI protein function. Thus, we hypothesize that the kinesin-associated protein, KAP3, as well as the kinesin-2 motors, KIF3A, KIF3B and KIF17, are essential for ciliary trafficking of GLI proteins, and that this transport is necessary or proper GLI processing and function. The rationale for this proposal is that a deeper mechanistic understanding of the ciliary trafficking of GLI proteins will significantly inform the development f novel therapies for a growing number of ciliopathies and HH-driven pathologies where de-regulated HH signaling promotes disease initiation and progression. To test our hypothesis, we propose two specific aims. In Aim 1 we will: 1) define the physical interactions of GLI proteins with the heterotrimeric kinesin-2 motor complex, 2) determine the role of heterotrimeric kinesin-2 motors in regulating the subcellular localization and processing of GLI proteins, and 3) investigate the functional consequences of disrupting KIF3-KAP3-GLI interactions on HH signal transduction. In Aim 2 we will: 1) define novel interactions between GLI proteins and the homodimeric kinesin-2 motor, KIF17, 2) elucidate the consequences of disrupting GLI-KIF17 interactions on GLI protein localization, processing and activity, and 3) investigate a novel role for KIF17 in GLI-mediated cerebellar progenitor proliferation in vivo. The proposed research will utilize a combination of in vitro (biochemical, imaging, and cell signaling assays) and in vivo (mouse genetics and chicken in ovo electroporations) approaches to define how kinesin-2 interactions with GLI proteins affects HH pathway function. This work will define new mechanisms that regulate HH pathway function, and will significantly impact our understanding of HH-driven developmental diseases and cancers.